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Enzymatic generation of peptides flanked by basic amino acids to obtain MS/MS spectra with 2× sequence coverage

Ebhardt, H Alexander; Nan, Jie LU ; Chaulk, Steven G; Fahlman, Richard P and Aebersold, Ruedi (2014) In Rapid Communications in Mass Spectrometry 28(24). p.43-2735
Abstract

RATIONALE: Tandem mass (MS/MS) spectra generated by collision-induced dissociation (CID) typically lack redundant peptide sequence information in the form of e.g. b- and y-ion series due to frequent use of sequence-specific endopeptidases cleaving C- or N-terminal to Arg or Lys residues.

METHODS: Here we introduce arginyl-tRNA protein transferase (ATE, EC 2.3.2.8) for proteomics. ATE recognizes acidic amino acids or oxidized Cys at the N-terminus of a substrate peptide and conjugates an arginine from an aminoacylated tRNA(Arg) onto the N-terminus of the substrate peptide. This enzymatic reaction is carried out under physiological conditions and, in combination with Lys-C/Asp-N double digest, results in arginylated peptides with... (More)

RATIONALE: Tandem mass (MS/MS) spectra generated by collision-induced dissociation (CID) typically lack redundant peptide sequence information in the form of e.g. b- and y-ion series due to frequent use of sequence-specific endopeptidases cleaving C- or N-terminal to Arg or Lys residues.

METHODS: Here we introduce arginyl-tRNA protein transferase (ATE, EC 2.3.2.8) for proteomics. ATE recognizes acidic amino acids or oxidized Cys at the N-terminus of a substrate peptide and conjugates an arginine from an aminoacylated tRNA(Arg) onto the N-terminus of the substrate peptide. This enzymatic reaction is carried out under physiological conditions and, in combination with Lys-C/Asp-N double digest, results in arginylated peptides with basic amino acids on both termini.

RESULTS: We demonstrate that in vitro arginylation of peptides using yeast arginyl tRNA protein transferase 1 (yATE1) is a robust enzymatic reaction, specific to only modifying N-terminal acidic amino acids. Precursors originating from arginylated peptides generally have an increased protonation state compared with their non-arginylated forms. Furthermore, the product ion spectra of arginylated peptides show near complete 2× fragment ladders within the same MS/MS spectrum using commonly available electrospray ionization peptide fragmentation modes. Unexpectedly, arginylated peptides generate complete y- and c-ion series using electron transfer dissociation (ETD) despite having an internal proline residue.

CONCLUSIONS: We introduce a rapid enzymatic method to generate peptides flanked on either terminus by basic amino acids, resulting in a rich, redundant MS/MS fragment pattern.

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Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
keywords
Amino Acids, Basic, Aminoacyltransferases, Peptides, Sequence Analysis, Protein, Tandem Mass Spectrometry
in
Rapid Communications in Mass Spectrometry
volume
28
issue
24
pages
9 pages
publisher
John Wiley & Sons
external identifiers
  • Scopus:84954445952
ISSN
1097-0231
DOI
10.1002/rcm.7069
language
English
LU publication?
yes
id
bc76bb03-e834-4ba6-abdb-afe1a0776e81
date added to LUP
2016-09-07 22:49:37
date last changed
2016-10-13 05:13:17
@misc{bc76bb03-e834-4ba6-abdb-afe1a0776e81,
  abstract     = {<p>RATIONALE: Tandem mass (MS/MS) spectra generated by collision-induced dissociation (CID) typically lack redundant peptide sequence information in the form of e.g. b- and y-ion series due to frequent use of sequence-specific endopeptidases cleaving C- or N-terminal to Arg or Lys residues.</p><p>METHODS: Here we introduce arginyl-tRNA protein transferase (ATE, EC 2.3.2.8) for proteomics. ATE recognizes acidic amino acids or oxidized Cys at the N-terminus of a substrate peptide and conjugates an arginine from an aminoacylated tRNA(Arg) onto the N-terminus of the substrate peptide. This enzymatic reaction is carried out under physiological conditions and, in combination with Lys-C/Asp-N double digest, results in arginylated peptides with basic amino acids on both termini.</p><p>RESULTS: We demonstrate that in vitro arginylation of peptides using yeast arginyl tRNA protein transferase 1 (yATE1) is a robust enzymatic reaction, specific to only modifying N-terminal acidic amino acids. Precursors originating from arginylated peptides generally have an increased protonation state compared with their non-arginylated forms. Furthermore, the product ion spectra of arginylated peptides show near complete 2× fragment ladders within the same MS/MS spectrum using commonly available electrospray ionization peptide fragmentation modes. Unexpectedly, arginylated peptides generate complete y- and c-ion series using electron transfer dissociation (ETD) despite having an internal proline residue.</p><p>CONCLUSIONS: We introduce a rapid enzymatic method to generate peptides flanked on either terminus by basic amino acids, resulting in a rich, redundant MS/MS fragment pattern.</p>},
  author       = {Ebhardt, H Alexander and Nan, Jie and Chaulk, Steven G and Fahlman, Richard P and Aebersold, Ruedi},
  issn         = {1097-0231},
  keyword      = {Amino Acids, Basic,Aminoacyltransferases,Peptides,Sequence Analysis, Protein,Tandem Mass Spectrometry},
  language     = {eng},
  month        = {12},
  number       = {24},
  pages        = {43--2735},
  publisher    = {ARRAY(0xa5e3a50)},
  series       = {Rapid Communications in Mass Spectrometry},
  title        = {Enzymatic generation of peptides flanked by basic amino acids to obtain MS/MS spectra with 2× sequence coverage},
  url          = {http://dx.doi.org/10.1002/rcm.7069},
  volume       = {28},
  year         = {2014},
}